Project Summary/Abstract Survival rates (app. 30 %) of acute myeloid leukemia (AML) have not been improved over 4 decades, except in some specialized instances. The long term aim of this study is to increase the cure rates of AML through clinical implementation of targeting a new cellular survival mechanism, i.e. mitochondrial (mt) unfolded protein response (mtUPR). We are proposing to conduct a clinical Phase 1/2 trial of ONC201, a first-in-class imipridone and to confirm and further investigate the underlying novel mechanism of action (MOA). We discovered in extensive preclinical studies that ONC201 induces apoptosis in AML but not in normal cells. Importantly, ONC201 has great efficacy in p53-mutated AML, the most chemotherapy-resistant subset, as well as in p53 wild-type AML. Our preclinical studies further demonstrate that ONC201 eliminates functionally- defined leukemia stem cells in patient-derived xenografts. Early trials initiated at MD Anderson show excellent tolerability of ONC201, micromolar plasma concentrations, and early clinical responses. We previously reported that ONC201 induces apoptosis mediated by the transcription factor ATF4, a hallmark of integrated stress response (ISR). However, ONC201 did not induce all characteristic molecular changes associated with classical ISRs (e.g., ER stress), suggesting an atypical MOA to induce ATF4. As break through progress reported in this re-submission, we have discovered that ONC201 directly binds and activates the mitochondrial protease, ClpP, resulting in selective mitochondrial proteolysis. The resultant reduction of mt protein pools induces so-called mt protein folding stress (mtPFS) and the protective transcriptional response against mtPFS termed mt unfolded protein response (mtUPR). Importantly, ATF4 is known to be induced through mtUPR, connecting our previous findings on ATF4 in a way different from classical ISRs. We here hypothesize that AML progenitor and stem cells are more susceptible to mtPFS than normal cells, and that ONC201 is targeting a novel point of vulnerability in AML pathobiology. The proposed clinical trial in leukemia provides a unique opportunity to thoroughly investigate this hypothesis. We will conduct a Phase 1/2 study of ONC201 in AML (Aim 1), and evaluate the underlying MOA (Aim 2). The Phase 1 trial will determine the safety and preliminary efficacy of ONC201 and Phase 2 the overall response rate. Changes in ATF4, mtUPR effector proteins, mt function and biogenesis in AML cells will be investigated using standard immunoblot and PCR methods as well as novel tools including CyTOF (single cell proteomics). We will also determine if ClpP, ATF4 and mtUPR effector proteins are potential biomarkers of clinical response to ONC201. Changes in clonal architecture will be monitored by flow cytometry and single-cell DNA sequencing. Genome-wide RNAseq will also be performed to further elucidate MOA and potential resistance. We expect these studies, which are at the cutting edge of our evolving knowledge of mitochondrial pathophysiology, to be developed into a highly effective and novel concept for the treatment of AML.